The present invention relates to a matrix suitable, inter alia, for the support, retention, and/or growth of living cells in a form suitable, for example, for cell transplantation. More particularly, it pertains to a reconstituted collagen matrix suitable for cell growth, such as chondrocyte cell growth for use in chondrocyte cell transplantation.
Injuries to the cartilage of the knee or other joints often result from abnormal mechanical loads which deform the tissue matrix. The loads applied to the joint can rupture the collagen network in the matrix and decrease the stiffness of the tissue.
Cartilage injuries are difficult to treat because articular cartilage has a limited capacity for regeneration once it has been damaged. Type II collagen is the main structural protein of the extracellular matrix in articular cartilage. Type II collagen, similar to other types of collagen, is comprised of three collagen polypeptides which form a triple helix configuration. The polypeptides are intertwined with each other and possess at each end telopeptide regions which provide the crosslinking between the collagen polypeptides. Collagen matrices in their natural state contain numerous crosslinked triple helices and the individual molecules have a molecular weight of about 300,000 daltons. Type II collagen is found almost exclusively in animal cartilage, while other types of collagen are found in animal hides, membranes, and bones.
Excessive degradation of Type II collagen in the outer layers of articular surfaces of joints is also caused by osteoarthritis. The collagen network is accordingly weakened and subsequently develops fibrillation whereby matrix substances, such as proteoglycans, are lost and eventually displaced entirely. Such fibrillation of weakened osteoarthritic cartilage can reach down to the calcified cartilage and into the subchondral bone (Kempson, G. E. et al., Biochim. Biophys. Acta 1976, 428, 741; Roth, V. and Mow, V. C., J. Bone Joint Surgery, 1980, 62A, 1102; Woo, S. L. -Y. et al., in Handbook of Bioengineering (R. Skalak and S. Chien Eds), McGraw-Hill, New York, 1987, pp. 4.1-4.44).
A method for regeneration-treatment of cartilage would be useful for treating arthritis and other joint conditions and could be performed at an earlier stage of joint damage, thus reducing the number of patients needing more extensive procedures, such as artificial joint replacement surgery. With such preventive methods of treatment, the number of patients developing osteoarthritis would also decrease.
Methods for growing and using chondrocyte cells are described by Brittberg, M. et al. (New Engl. J. Med. 1994, 331, 889). Autologous transplants using cells grown with these methods are also disclosed. Additionally, Kolettas et al. examined the expression of cartilage-specific molecules, such as collagens and proteoglycans, under prolonged cell culturing (J. Cell Science 1995, 108, 1991). They found that, despite morphological changes during culturing in monolayer cultures (Aulthouse, A. et al., In Vitro Cell Dev. Biol., 1989, 25, 659; Archer, C. et al., J. Cell Sci. 1990, 97, 361; Hxc3xa4nselmann, H. et al., J. Cell Sci. 1994, 107, 17; Bonaventure, J. et al., Exp. Cell Res. 1994, 212, 97), when compared to suspension cultures grown over agarose gels, alginate beads or as spinner cultures (retaining a round cell morphology) tested by various scientists, such morphologies did not change the chondrocytexe2x80x94expressed markers such as types II and IX collagens and the large aggregating proteoglycans, aggrecan, versican and link protein did not change (Kolettas, E. et al., J. Cell Science 1995, 108, 1991).
In addition, chondrocyte cells from donors have been grown in vitro to form neocartilage which has been implanted into animals (Adkisson et al., xe2x80x9cA Novel Scaffold-Independent Neocartilage Graft for Articular Cartilage Repair,xe2x80x9d ICRS 2nd Symposium International Cartilage Repair Society, Nov. 16-18, 1998). Further, chondrocyte cells have been seeded onto the cartilage surface of osteochondral cores to attempt cartilage regeneration (Albrecht et al., xe2x80x9cCircumferential Seeding of Chondrocytes: Towards Enhancement of Integrative Cartilage Repair,xe2x80x9d ICRS 2nd Symposium International Cartilage Repair Society, Nov. 16-18, 1998). Articular surface defects in knee joints have been treated with various cultured chondrocytes (Stone et al., Operative Techniques in Orthopaedics 7(4), pp. 305-311, October 1997 and Minas et al., Operative Techniques in Orthopaedics 7(4), pp. 323-333, October 1997).
Membranes and certain of their general properties are described in the following references:
U.S. Pat. No. 5,837,278xe2x80x94Geistlich et al. describe a collagen-containing membrane which is resorbable and is used in guided tissue regeneration. The membrane has a fibrous face which allows cell growth thereon and a smooth face opposite the fibrous face which inhibits cell adhesion thereon. The membrane product is derived from a natural collagen membrane (from the hide or tendons of calves or piglets) and, although treated, it is described as maintaining its natural structural features. The collagen is purified with alkaline agents to defat the collagen and degrade substances, and then the purified collagen is acidified, washed, dried, degreased, and optionally crosslinked. The fats are saponified. The membrane is described as containing about 95% by weight native collagen.
PCT WO 96/25961xe2x80x94Geistlich et al. describe a matrix for reconstructing cartilage tissue which consists of Type II collagen, optionally including crosslinking. In producing the matrix, cartilage is taken from an animal and frozen, subjected to size reduction, dewatered, defatted, washed, and treated with alkaline materials. Non-collagen alkaline soluble proteins are denatured, destroyed, dissolved, and eliminated. Dialysis and freeze drying are mentioned as possible treatment steps. The matrix material is stamped to form a required shape and then it is sterilized.
U.S. Pat. No. 4,424,208xe2x80x94Wallace et al. describe an injectable collagen implant material comprising particulate crosslinked atelopeptide collagen and reconstituted atelopeptide collagen fibers dispersed in an aqueous carrier. The atelopeptide form of collagen lacks the native telopeptide crosslinking. In the method described in the ""208 patent, collagen obtained from bovine or porcine corium (sub-epithelial skin layer) is softened by soaking in a mild acid; depiliated; comminuted by physical treatment, such as grinding; solubilized by treatment with acid and a proteolytic enzyme; treated with an alkaline solution; and freed of enzyme. The crosslinked gel form of collagen is formed by radiation-induced or chemical-induced crosslinking, such as by addition of glutaraldehyde. Meanwhile, the fibrous form of collagen is produced by neutralizing the solution with a buffer, such as Na2HPO4. The collagen content of the injectable implant comprises 5-30% fibrous collagen and 70-98% of the crosslinked gel form of collagen.
U.S. Pat. No. 4,488,911xe2x80x94Luck et al. describe the formation of collagen fibers free of the immunogenic, telopeptide portion of native collagen. The telopeptide region provides points of crosslinking in native collagen. The fibers, which may be crosslinked, are described for use as sponges, prosthetic devices, films, membranes, and sutures. In the method described in the ""911 patent, (non-Type II; Type I and others) collagen obtained from tendons, skin, and connective tissue of animals, such as a cow, is dispersed in an acetic acid solution, passed through a meat chopper, treated with pepsin to cleave the telopeptides and solubilize the collagen, precipitated, dialyzed, crosslinked by addition of formaldehyde, sterilized, and lyophilized. The ""911 patent indicates that its disclosed method obtains the atelocollagen form of collagen, free from noncollagen proteins, such as glycosaminoglycans and lipids. Further, it describes that the collagen may be used as a gel to make, for example, a membrane, film, or sponge and that the degree of crosslinking of the collagen can be controlled to alter its structural properties.
Notwithstanding the foregoing disclosures, applicants believe that there remains a need for a satisfactory and effective scaffold for cell growth, particularly chondrocyte cell growth.
The present invention provides a reconstituted biocompatible, resorbable Type II collagen-based matrix for use, for example, as a scaffold on which cells, such as chondrocyte cells, can be cultured, and a method for producing the matrix. Furthermore, the matrix can be used as a sheet to cover small cartilage lesions or can be used in combination with cells other than chondrocytes (i.e., with mesenchymal stem cells). The Type II collagen of the matrix of the present invention is solubilized by breaking the crosslinking of the collagen through its telopeptide region into its non-crosslinked atelocollagen form. The matrix may be a support matrix which provides a scaffold on which cells can be grown and retained.
In one embodiment of the present invention, the matrix is produced by the reconstitution of animal cartilage tissue (horse, pig, cow (or calf), goat, chicken, or kangaroo) from the solubilization of the Type II collagen and removal of associated proteins and molecules from the tissue and the subsequent isolation, crosslinking, and lyophilization to form a fleece-like matrix.
In another embodiment, the matrix composition is formed from recombinantly produced Type II collagen which is solubilized and crosslinked to form a fleece-like matrix.
In still another embodiment, the matrix composition is used as a base for growing and attaching living cells, particularly chondrocyte cells, thereon. In this embodiment, chondrocytes cells adhere to the matrix and grow, substantially as they do in vivo.
In a further embodiment, the present invention provides an implantable article including chondrocyte cells retained on the support matrix and a method of making the same. In another embodiment, the present invention provides a method for tissue repair, particularly the effective treatment of articulating joint surface cartilage in an animal by the transplantation of an implantable article including chondrocyte cells, either autologous or homologous, retained on or combined with the support matrix.